Downregulation of the calcium-sensing receptor in vascular endothelial cells from zucker diabetic fatty (ZDF) rats

1Absi, M., 1Weston, A.H., 1Ward, D.T., 2Dodd, R.H., 3Ruat, M. & 1Edwards, G. 1Faculty of Life Sciences, University of Manchester, M13 9PT, UK; 2Institut de Chimie des Substances Naturelles, UPR2301, CNRS, 91198 Gif-sur-Yvette, France ; 3UPR9040, Institut de Neurobiologie Alfred Fessard, IFR 2118 CNRS, 91198 Gif-sur-Yvette, France.

Bukoski and coworkers (1997) concluded that extracellular calcium-induced relaxation of rat mesenteric arteries was due to activation of calcium-sensing receptors (CaSRs) located on perivascular nerves. However, using the same preparation, our experiments (Weston et al., 2005) have provided evidence for endothelial cell CaSRs which initiate endothelium-dependent smooth muscle hyperpolarization (EDH). The aim of the present study was to investigate this phenomenon in mesenteric arteries from rats with diabetes (a condition which is associated with endothelial cell dysfunction; reviewed by Triggle et al., 2004).

Experiments were performed on 2nd and 3rd order mesenteric artery branches from 12-14 weeks old male Zucker diabetic fatty (ZDF) rats or age-matched lean controls (ZL) previously killed by stunning and cervical dislocation. Membrane potential (m.p.) was recorded using sharp micro-electrodes (see Weston et al., 2002 ). Results, expressed as mean±SEM, were analysed by 2-way ANOVA and Tukey-Kramer tests.

The myocyte m.p. of endothelium-intact arteries was similar in control and ZDF rats (-53.9±0.3mV, n=12 and -53.9±0.3mV, n=12, respectively). EDH induced by calindol, a positive allosteric modulator of CaSR (Petrel et al., 2004), was concentration-dependent in arteries from control and ZDF rats, but was significantly smaller in the latter. Thus, 300 nM calindol hyperpolarized the myocytes by 12.1±0.9mV (n=8) in control arteries and 9.2±0.9mV (n=8) in ZDF arteries, but in both the response was totally abolished by 10µM TRAM-34 (an IKCa inhibitor) suggesting that the response to calindol was due to the opening of endothelial IKCa. However, there was no significant difference between the EDH induced by the IKCa opener 1-ethyl-2-benzimidazolinone (300 µM) in ZDF and ZL arteries (14.5±1.4 mV and 12.1±1.1mV, respectively, each n=4). Our initial conclusion, that expression of CaSR was reduced in the ZDF arteries, was supported by Western blot analysis in which bands produced by artery membrane preparations from ZDF rats (matched for protein content) were consistently fainter than those from ZL rats (each n=3).

This study thus provides evidence for decreased expression of CaSRs on mesenteric artery endothelial cells in a rat model of diabetes. Since endothelial cell CaSR channels are basally active in normal endothelial cells under quasiphysiological in vitro conditions (Weston et al., 2005), we speculate that loss of a such a (putative) relaxant pathway involving the CaSR and endothelial IKCa channels may contribute to the hypertension associated with type 2 diabetes.

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Supported by the British Heart Foundation.